Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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The DSIF subunits Spt4 and Spt5 have several unexpected, distinct roles in regulating immunoglobulin class switch recombination

ABSTRACT: Class-switch recombination (CSR), induced by activation-induced cytidine deaminase, can be divided into two phases: DNA cleavage of the switch (S) regions, and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in CH12F3-2A cells, and Spt4 and Spt5, carry out independent functions in CSR. Depleting Spt5 reduced the H3K4me3 levels and DNA cleavage at the S? region, whereas Spt4 knockdown did not perturb the H3K4me3 status or S region cleavage. Thus, we conclude that Spt5 plays a role similar to the histone chaperone FACT by regulating histone modification and DNA cleavage in CSR. Assay systems using synthetic repair substrates revealed that both Spt4 and Spt5 are required for non-homologous end joining and homologous recombination. Taken together, Spt4 and Spt5 are involved in multiple CSR steps, and can function independently. Spt4 or Spt5 were knocked down in CH12F3-2A cells in the presence of CD40L, IL4, TGFb (CIT) stimulation. RNA from each samples were extracted and relative difference in transcript level were compared with control RNAi-treated, CIT-stimulated samples.

ORGANISM(S): Mus musculus

SUBMITTER: Satoshi Kondo

PROVIDER: E-GEOD-33206 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

The DSIF subunits Spt4 and Spt5 have distinct roles at various phases of immunoglobulin class switch recombination.

Stanlie Andre A Begum Nasim A NA Akiyama Hideo H Honjo Tasuku T

PLoS genetics 20120426 4

Class-switch recombination (CSR), induced by activation-induced cytidine deaminase (AID), can be divided into two phases: DNA cleavage of the switch (S) regions and the joining of the cleaved ends of the different S regions. Here, we show that the DSIF complex (Spt4 and Spt5), a transcription elongation factor, is required for CSR in a switch-proficient B cell line CH12F3-2A cells, and Spt4 and Spt5 carry out independent functions in CSR. While neither Spt4 nor Spt5 is required for transcription ...[more]

PMID: 22570620

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Project description:The Spt4-Spt5 complex is conserved and essential RNA polymerase elongation factor. To investigate the role of the Spt4-Spt5 complex in non-coding transcription during development, we used the unicellular model Paramecium tetraurelia. In this organism harboring both germline (micronucleus - MIC) and somatic nuclei (macronucleus - MAC), massive transcription of the entire germline genome takes place during meiosis. This phenomenon starts a series of events mediated by different classes of non-coding RNAs that control developmentally programmed DNA elimination. We focused our study on Spt4, a small zinc-finger protein encoded in P. tetraurelia by two genes expressed constitutively and two genes expressed during meiosis. SPT4 genes are not essential in vegetative growth, but they are indispensable for sexual reproduction, even though genes from both expression families show functional redundancy. As we were mostly interested in transcription of the germline genome in the MIC during meiosis, we decided to probe the composition of the Spt5-Spt4 complex characteristic to this nucleus. Experiments in which Spt4mB-3xFLAG was used as bait demonstrated its interaction with either Spt5m or Spt5v, which is consistent with the Spt4mB-GFP localization pattern. Interestingly, mass spectrometry revealed that Spt5m-3xFLAG (which is present only in the MIC at the analyzed stage) interacts either with the meiotic Spt4mB or with Spt4vA. Silencing of the SPT4 genes resulted in the absence of double-stranded ncRNAs and reduced levels of scnRNAs – 25 nt-long sRNAs produced from these double-stranded precursors in the germline nucleus. Moreover, we observed that the presence of a germline-specific Spt4-Spt5m complex is necessary for transfer of the scnRNA-binding PIWI protein between the germline and somatic nucleus. Our study establishes that Spt4, together with Spt5m, is essential for expression of the germline genome and necessary for developmental genome rearrangements.

Project description:Classical non-homologous end-joining (C-NHEJ) is a major mammalian DNA double strand break (DSB) repair pathway. Core C-NHEJ factors, such as XRCC4, are required for joining DSB intermediates of the G1 phase-specific V(D)J recombination reaction in progenitor lymphocytes. Core factors also contribute to joining DSBs in cycling mature B-lineage cells, including DSBs generated during antibody class switch recombination (CSR) and DSBs generated by ionizing radiation (IR). The XLF C-NHEJ factor is dispensable for V(D)J recombination in normal cells, but, due to functional redundancy, is absolutely required for this process in cells deficient for the ATM DSB response factor. The recently identified PAralogue of XRCC4 and XLF (PAXX) factor has homology to these two proteins and variably contributes to IR-induced DSB repair in human and chicken cells. We now report that PAXX is dispensable for joining V(D)J recombination DSBs in G1-arrested mouse pro-B cell lines, dispensable for joining CSR-associated DSBs in a cycling mouse B cell line, and dispensable for normal IR-resistance in both G1-arrested and cycling pro-B lines. However, we find that combined deficiency for PAXX and XLF in G1-arrested pro-B lines abrogates DSB joining during V(D)J recombination and sensitizes the cells to IR exposure. Thus, PAXX provides core C-NHEJ factor-associated functions in the absence of XLF and vice versa in G1-arrested Pro-B cell lines. Finally, we also find that PAXX-deficiency has no impact on V(D)J recombination DSB joining in ATM-deficient pro-B lines. We discuss implications of these findings with respect to potential PAXX and XLF functions in C-NHEJ. Examination of CSR Switch mu-to-alpha junctions from mu bait DSBs using LAM-HTGTS and Illumina Miseq. Two clones of PAXX-/- and XLF-/- and 1 clone of Ligase4-/- were derived from the parental CH12F3 line; the second Ligase4-/- clone was acquired from Keifei Yu. Two clones of XLF-/-PAXX-/-CH12 cells were derived from one of the PAXX-/- clones. Three biological replicates were analyzed for each clone.

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Dataset Information

The DSIF subunits Spt4 and Spt5 have several unexpected, distinct roles in regulating immunoglobulin class switch recombination

Publications

The DSIF subunits Spt4 and Spt5 have distinct roles at various phases of immunoglobulin class switch recombination.

Similar Datasets

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